Journal of Neuroscience Research 23:274-281 (1989) Induction of Morphological Differentiation of Human Neuroblastoma Cells Is Accompanied by Induction of Tissue-Type Plasminogen Activator zyxw T. Neuman, R.W. Stephens, E.-M. Salonen, T. Timmusk, and A. Vaheri Department of Virology, University of Helsinki, Helsinki, Finland (T.N., R.W.S., E.-M.S., A.V.), and Department of Molecular Genetics, Institute of Chemical Physics and Biophysics; Estonian Academy of Sciences, Tallinn, Estonian SSR, Union of Soviet Socialist Republics (T.N., T.T.) Human SH-SY5Y neuroblastoma cells treated with retinoic acid, zyxwvuts 12-0-tetradecanoyl-phorbol-13-acetate (TPA) or nerve growth factor differentiated morpho- logically to neuronlike cells with increased amounts of neurofilament protein and mRNA. All three effectors induced an increase in the amount of relative molec- ular weight (M,) 70 000 tissue-type plasminogen ac- tivator (t-PA) and its mRNA, as determined by im- munocapture, enzyme activity, and Northern blotting analyses. About 90% of the t-PA activity was secreted to the culture medium. In contrast, of the three ef- fectors studied, only TPA induced transcription of the proto-oncogene c-fos, studied as a control gene responsive to various stimuli, and induced a rapid increase in urokinase-type PA (u-PA). Most of the u-PA activity induced by TPA remained cell-associ- ated. Because induction of differentiation correlated closely with induction of t-PA, and not u-PA, the au- thors propose that t-PA may have a functional role in the morphological differentiation of neuronal cells. Key words: differentiation, neuroblastoma, tissue- type plasminogen activator INTRODUCTION Plasminogen activation (PA) is by far the most im- portant mechanism of extracellular proteolysis (Dan@ et al., 1985). It is a cascade-like process, regulated at several levels. Cells synthesize plasminogen activators as inac- tive proenzymes; these can be activated experimentally by minute amounts of plasmin to active two-chain forms. This mechanism of activation is, however, possibly not the natural one, as it would require the end product of the cascade-plasmin-to be present for the first steps of regulation. The two types of plasminogen activators- urokinase-type (u-PA) and tissue-type (t-PA)-are dis- tinct molecules and carry distinct functions in vivo; u-PA is known to bind to a specific receptor molecule present in the plasma membrane of cells; this binding is mediated by a specific region in the epidermal growth factor-like domain of the u-PA molecule (Blasi, 1988). These two types of activators have powerful inhibitors, of which several different types have been characterized already (Sprengers and Kluft, 1987). They include PAI-1 (pre- viously known as endothelial inhibitor) relative molecular weight (M,) 52 000), PAI-2 (placental or mono- cyte/macrophage-derived inhibitor), PAI-3 (present in urine), and protease nexin, which is not a specific PA inhibitor but inhibits also thrombin and plasmin. The only well-characterized substrate for both u- PA and t-PA is plasminogen (Dan@ et al., 1985; Blasi, 1988), a proenzyme that is widely distributed in practi- cally all body fluids and tissues. PA converts plasmino- gen to active plasmin, a serine proteinase with a broad spectrum of target proteins. These include not only fibrin and matrix proteins such as fibronectin, but also certain latent enzymes, e.g., interstitial and basement membrane procollagenase and latent macrophage elastase, involved in degradation of tissue matrices. Thus, activation of plasminogen can trigger a set of enzymes, leading to complete tissue matrix destruction. Plasmin, too, has powerful inhibitors present in body fluids (Travis and Salvesen, 1983). Of these, the most important are the fast-acting alpha-2-antiplasmin and alpha-2-macroglob- ulin. PA production has been associated with invasive tumor cells (Dan0 et al., 1985) and with certain types of normal cells engaged in cell migration and penetration Received October 20, 1988; revised January 25, 1989; accepted Feb- ruary 1. 1989. Address reprint requests to Dr. Antti Vaheri, Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki, Fin- land. zyxwvu 0 1989 Alan R. Liss, Inc.